In the past year, we have advanced the objectives of this project along several lines. (1) Development of a reverse genetics system. Three years ago, Komoto et al (2006) described a helper virus-dependent, single-gene replacement reverse genetics system for rotavirus. This system used a replication-deficient vaccinia virus (rDIs-T7pol) as the source of T7 RNA polymerase (T7pol) to drive the transcription of a SA11 rotavirus VP4 gene from a transfected cDNA plasmid. Infection of the transfected cells with a helper rotavirus (KU strain) allowed for the recovery of recombinant viruses that had incorporated the SA11 VP4 gene into the KU genome. Shortly thereafter, Kobayashi et al (2007) reported the development of a fully plasmid-based reverse genetic system for reovirus, another segmented dsRNA virus in the Reoviridae family. In this system, 10 plasmids, each of which contains the cDNA of an authentic reovirus gene behind a T7 promoter and upstream of an HDV ribozyme, are transfected into murine cells (L929) infected with rDIs-T7pol. Viral transcripts made from the cDNAs launch the production of recombinant infectious virus, with yields reaching 10 to 100 PFU per transfected 60-mm well (10ex6 cells). We have been successful in developing a helper virus-dependent, single-gene replacement reverse genetics system for rotavirus using an approach reminiscent of that described by Komoto et al (2006). The design and efficiency of the system is such that is can be used to modify many if not all of the rotaviruses genes. We have continued attempts to establish a fully plasmid-based reverse genetics system to rotavirus. As part of that effort, we have set up and successfully used the plasmid-based reovirus reverse genetics system. (2) In previous studies, we showed that rotavirus subverts IFN signaling by the action of its nonstructural protein NSP1. Specifically, we learned that NSP1 has the potential to interact with multiple members of the IRF family of proteins. This interaction is correlated with the degradation of IRF protein (e.g., IRF3, IRF5, and IRF7) in infected cells, via a proteasome-dependent process. This degradation prevents rotavirus-infected cells from expressing IFN. In recent studies, we found that the ability of rotavirus NSP1 to degrade IFN-signaling targets varies among different virus strains. For example, the NSP1 of some virus strains were shown to degrade three IRF proteins: IRF3, IRF5, and IRF7 (si SA11-4F, si RRV, mu ETD, po Gottfried);others two IRF proteins: IRF3 and IRF7 (bo UK) or IRF3 and IRF5 (la 30-96). At least one other virus (po OSU) failed to degrade any of these IRF proteins, but was capable of degrading beta-TRCP, a factor necessary for the activation of NF-kB. Moreover, we found that individual types of NSP1 may vary in their ability to degrade an IFN-signaling target as a factor of the species origin of the target. For example, NSP1 of the bo UK virus can degrade simian IRF3, but not human IRF3. Finally, we found that the type of cell used in NSP1 functional assays also impacts the ability of NSP1 to degrade an IFN-signaling target. As an example, transfection experiments have shown that NSP1 of po Gottfried rotavirus can degrade hu IRF3 expressed in human and simian cells, but not in porcine cells. The differences noted for the activities of NSP1 are perhaps not surprising given the remarkable sequence variation of the protein among different virus strains. (3) Analysis of the diversity and evolution of the rotavirus genome. The scarcity of complete genome sequence information for the rotaviruses prevents a comprehensive molecular analysis of rotavirus diversity and evolution, limits our capacity to assess the impact of rotavirus vaccines on the genetic make-up of viruses circulating in the human population, and undermines the full potential of reverse genetics systems. To address the need for additional sequence information, we have initiated several large scale genomic sequencing projects (a)-(c). (a) An important question is whether the widespread use of the RotaTeq and Rotarix vaccines will induce antigenic and/or genetic changes in commonly circulating rotaviruses or will lead to the emergence of new G-type strains not covered by the vaccines. To address this question, we initiated a project to sequence the complete genomes of rotaviruses in stool samples that were collected from 160 sick children at Vanderbilt Medical Center (Nashville, TN) during the 2005-06 to 2008-09 winter seasons. The four seasons covered by these samples are particularly significant as they include seasons prior to and after the RotaTeq vaccine was introduced into the Nashville community. The sequence data will be examined for evidence that vaccine usage induces shifts in the equilibrium of co-circulating G/P-type viruses and changes to the antigenic epitopes of the VP4 and VP7 proteins of circulating strains. (b) To gain insight into the diversity and evolution of human rotaviruses circulating in a single location over a long period of time, we sequenced 51 G3P8 rotaviruses isolated from sick children during 1974 to 1991 at Childrens Hospital National Medical Center, Washington DC. The results from this study revealed for the first time that genetically-distinct rotavirus clades of the same G/P-type, but with varying neutralization epitopes, can co-circulate and cause disease. The findings also indicate that although genome reassortment can occur among RVs, most reassortant strains are replaced overtime by lineages with preferred gene constellations. Thus, the G3P8 population is dynamic and in flux, in part driven by the competing pressures to maintain ideal gene constellations while increasing genetic diversity via reassortment. (c) Dr. Albert Kapikian and colleagues in the LID developed a multivalent rotavirus vaccine through reassortment of a panel of antigenically-distinct human rotaviruses with a bovine rotavirus (UK BRV vaccine). This live attenuated virus vaccine includes six reassorts differing in the serotype (G1, G2, G3, G4, G8, or G9) of their outer capsid component VP7. Master seed lots were prepared for each reassortant by the NIH (NIH Master Seeds), and then distributed to several companies interested in producing rotavirus vaccines at low cost in developing counties. As part of their program to develop the vaccine, one company has generated their own Master Seeds (Shantha) from the NIH Master Seeds, and then passed their Master Seed subsequently to produce Working Batches and Production Batches of each viral component of the UK BRV vaccine. To support the production and widespread use of low-cost effective rotavirus vaccines, we have agreed to sequence the genomes of the Master Seeds and the Working and Production Batches generated by the companies. This information is necessary to provide safety information concerning the genetic stability of the virus components during vaccine production. In the past year, we sequenced 13 complete genomes and parts of 11 other genomes of the viruses in the Master Seeds or Working or Production Batches. In total, 176 viral genes were sequenced. GenBank accession numbers were obtained for all sequenced genes. This information is being shared with all the vaccine producers to assist in seeking regulatory approval for using the vaccine.